Methods
Subjects . COVID-19 patient blood samples used for immunophenotyping were obtained during patients’ visit or hospitalizations at SUNY Downstate Medical Center in New York from May through to July 2020. Patients were defined as 1) non-hospitalized, with and without presentation of COVID-19 symptoms and 2) hospitalized with presentation of COVID-19 symptoms. Peripheral blood from venipuncture was drawn into EDTA and Heparin coated vacutainer tubes for immunophenotyping and processed within 48h of blood draw. Control blood samples from healthy volunteers without SARS-CoV-2 infection or co-morbid conditions were collected after obtaining written informed consent.
Flow Cytometry . Whole blood was stained per the clinical standard immunophenotyping protocols (Amerimmune LLC, Fairfax, VA). The samples were stained with the multiple antibody combinations for 30 minutes at 4˚C. RBCs were lysed using BD FACS lysis solution (BD Bioscience, San Jose, CA) as per manufacture directions. In brief, freshly obtain peripheral blood mononuclear cells (PBMC) were separated from 2 mL of whole blood within 24h of collection and diluted 1:1 with phosphate buffered saline pH 7.2 (PBS) (Thermo Fisher Scientific, Carlsbad, CA) using Lymphoprep (Stem cell Technologies, Cambridge, MA) and Accuspin tubes (Sigma-Aldrich, St. Louis, MO) as per manufactures directions. PBMCs’ were washed in PBS and resuspended in 0.5 mL PBS. 100 µL of the PBMCs were immunostained with a mixture of antibodies at 4℃ for 1 hour. Cells were washed and resuspended in PBS prior to acquisition. Antibodies used for the immune phenotyping of patient samples and gating strategy for active caspase-1 staining are detailed in the Supplementary information section of the manuscript ( Supplemental Table 1).
Apoptosis (caspase 3/7) and pyroptosis (caspase-1) were measured by flow cytometry using fluorescent-labeled inhibitors of caspase probe assay (FLICA; Immunochemistry Technologies, Minneapolis, MN). As a control, PBMCs were stimulated with nigericin for 2 hours. FAM-FLICA probes specific for caspase-1 or caspase 3/7 were added to 50 µl PBMC or whole blood and incubated for 1h at 37℃. Cells were subsequently washed and stained with a cocktail of fluorescently conjugated antibodies against CD45 PE-CY7 [HI30], CD3 AF700 [UCHT1], CD4 PE [RPA-T4], CD45RO PerCP-EF710 [UCHL1] and Viability Dye 780 (Thermo Fisher Scientific, Carlsbad, CA). Red blood cells were identified as CD235-positive cells in the experiments where caspase3/7 activity was measured. Samples were acquired on a 3 laser BD FACS Canto 10. CS&T beads (BD Bioscience, San Jose, CA) were acquired daily to ensure consistent performance of the BD FACS Canto 10 Canto10. The BD FACS Canto 10 utilized for this study has been validated for T, B, NK and Dendritic cell immunophenotyping clinical diagnostic testing. Denovo FCS Express v6 clinical edition (De Novo Software, Pasadena, CA) was used for flow cytometric analyses.
Plasma experiments . Plasma was separated from whole blood following centrifugation at 960 RCF. Cells (RBC and WBC) were either incubated at 37 °C alone or in the presence of trypsin for 1 hour then washed with 10 packed cell volumes of RPMI 1640 incomplete medium. Plasma was either held at room temperature (18 - 25°C) or heat inactivated at 56°C for 1 hr. Plasma was added back to the RBC/WBC in a 1:1 ratio and incubated overnight, rocking at room temperature. RBC caspase 3/7 activity was measured as described under flow cytometry.
Public SARS-CoV-2 and COVID-19 Transcriptome Analyses . Single cell RNA-Seq data from three COVID-19 participants that were ventilated and diagnosed with acute respiratory distress syndrome at 2-16 days after symptom onset and from 6 healthy controls was accessed from GEO (30). RNA-Seq data from cell lines infected in vitrowith SARS-CoV-2 was accessed from GEO: GSE147507 (31).Expression values for Caspase genes were normalized by DESeq2.
Ex vivo stimulation studies . Active caspase-1 in COVID-19 infected patient samples.  Whole blood from a COVID-19 positive patient was either (A) untreated or (B) treated with the pan-caspase inhibitor emricasan (Sigma Aldrich, MO, SML2227-5MG) or selective caspase-1 inhibitor VX765 overnight at 37 degrees in a water bath. Subsequently, PBMCs were purified (Accuspin System – Histopaque 1077; Sigma Aldrich, MO, A6929) and incubated with nigericin (Immunochemistry Technologies, MN) for 2h.  A Fam-FLICA probe specific for active caspase-1 was added to 50 µl PBMC, incubated for 1h at 37℃.  PMBCs were washed with cell wash buffer (Immunochemistry Technologies, MN) and stained with a cocktail of fluorescently conjugated antibodies against CD45 PE-CY7 [HI30], CD3 AF700 [UCHT1], CD4 PE [RPA-T4], CD45RO PerCP-EF710 [UCHL1] and Viability Dye 780 (Thermo Fisher Scientific, Carlsbad, CA).  Lymphocytes were identified using a standard gating schematic which incorporated gating of lymphocytes on an FSC/SSC plot and singlets on a FSC-A/FSC-H plot. Lymphocytes were further identified as CD45+ on a CD45/SSC plot and subsequent CD3-, CD3+ and CD3+CD4+ cells were identified on a CD45+ CD3/CD4 plot.
Statistical analysis . Demographic and HIV-related characteristics were described using the median, first quartile (Q1), and third quartile (Q3) for continuous variables and frequency for categorical variables. Differences among continuous variables were evaluated by either the Mann-Whitney, student t test or Krustal-Wallis test with Dunn’s multiple comparisons. Relationships among parameters were examined by Pearson correlation for continuous variables. All statistical tests were performed with GraphPad Prism version 8.0 (Graphpad Software Inc., CA, USA). Statistical significance is indicated as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. P-values ≤0.100, but not significant, are noted as statistical trends.
Study approval . All clinical investigations were conducted according to Declaration of Helsinki principles. All human studies were approved by institutional review boards (IRB 269846-10 and 1285028 protocols from State University of New York Downstate Medical Center and Amerimmune respectively). Written informed consent was received from participants prior to inclusion in the study.